Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. It is well known that complete IgG antibody molecules are Y-shaped molecules comprising four polypeptide chains: two heavy chains and two light chains. Each light chain consists of two domains, the N-terminal domain being known as the variable or VL domain (or region) and the C-terminal domain being known as the constant (or CL) domain (constant kappa (Cκ) or constant lambda (Cλ) domain). Each heavy chain consists of four or five domains, depending on the class of the antibody. The N-terminal domain is known as the variable (or VH) domain (or region), which is followed by the first constant (or CH1) domain, the hinge region, and then the second and third constant (or CH2 and CH3) domains. In an assembled antibody, the VL and VH domains associate together to form an antigen binding site. Also, the CL and CH1 domains associate together to keep one heavy chain associated with one light chain. The two heavy-light chain heterodimers associate together by interaction of the CH2 and CH3 domains and interaction between the hinge regions on the two heavy chains.
It is known that proteolytic digestion of an antibody can lead to the production of antibody fragments (Fab and Fab2). Such fragments of the whole antibody can exhibit antigen binding activity. Antibody fragments can also be produced recombinantly. Fv fragments, consisting only of the variable domains of the heavy and light chains associated with each other may be obtained. These Fv fragments are monovalent for antigen binding. Smaller fragments such as individual variable domains (domain antibodies or dABs; Ward et al., 1989, Nature 341(6242): 544-46), and individual complementarity determining regions or CDRs (Williams et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86(14): 5537-41) have also been shown to retain the binding characteristics of the parent antibody, although most naturally occurring antibodies generally need both a VH and VL to retain full binding potency.
Single chain variable fragment (scFv) constructs comprise a VH and a VL domain of an antibody contained in a single polypeptide chain wherein the domains are separated by a flexible linker of sufficient length (more than 12 amino acid residues), that forces intramolecular interaction, allowing self-assembly of the two domains into a functional epitope binding site (Bird et al., 1988, Science 242(4877): 423-26). These small proteins (MW ˜25,000 Da) generally retain specificity and affinity for their antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules.
An advantage of using antibody fragments rather than whole antibodies in diagnosis and therapy lies in their smaller size. They are likely to be less immunogenic than whole antibodies and more able to penetrate tissues. A disadvantage associated with the use of such fragments is that they have only one antigen binding site, leading to reduced avidity. In addition, due to their small size, they are cleared very fast from the serum, and hence display a short half-life.
It has been of interest to produce bispecific antibodies (BsAbs) that combine the antigen binding sites of two antibodies within a single molecule, and therefore, would be able to bind two different antigens simultaneously. Besides applications for diagnostic purposes, such molecules pave the way for new therapeutic applications, e.g., by redirecting potent effector systems to diseased areas (where cancerous cells often develop mechanisms to suppress normal immune responses triggered by monoclonal antibodies, like antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)), or by increasing neutralizing or stimulating activities of antibodies. This potential was recognized early on, leading to a number of approaches for obtaining such bispecific antibodies. Initial attempts to couple the binding specificities of two whole antibodies against different target antigens for therapeutic purposes utilized chemically fused heteroconjugate molecules (Staerz et al., 1985, Nature 314(6012): 628-31).
Bispecific antibodies were originally made by fusing two hybridomas, each capable of producing a different immunoglobulin (Milstein et al., 1983, Nature 305(5934): 537-40), but the complexity of species (up to ten different species) produced in cell culture made purification difficult and expensive (George et al., 1997, THE ANTIBODIES 4: 99-141 (Capra et al., ed., Harwood Academic Publishers)). Using this format, a mouse IgG2a and a rat IgG2b antibody were produced together in the same cell (e.g., either as a quadroma fusion of two hybridomas, or in engineered CHO cells). Because the light chains of each antibody associate preferentially with the heavy chains of their cognate species, three major species of antibody are assembled: the two parental antibodies, and a heterodimer of the two antibodies comprising one heavy/light chain pair of each, associating via their Fc portions. The desired heterodimer can be purified from this mixture because its binding properties to Protein A are different from those of the parental antibodies: rat IgG2b does not bind to Protein A, whereas the mouse IgG2a does. Consequently, the mouse-rat heterodimer binds to Protein A but elutes at a higher pH than the mouse IgG2a homodimer, and this makes selective purification of the bispecific heterodimer possible (Lindhofer et al., 1995, J. Immunol. 155(1): 219-25). The resulting bispecific heterodimer is fully non-human, hence highly immunogenic, which could have deleterious side effects (e.g., “HAMA” or “HARA” reactions), and/or neutralize the therapeutic. There remained a need for engineered bispecifics with superior properties that can be readily produced in high yield from mammalian cell culture.
Despite the promising results obtained using heteroconjugates or bispecific antibodies produced from cell fusions as cited above, several factors made them impractical for large scale therapeutic applications. Such factors include: rapid clearance of heteroconjugates in vivo, the laboratory intensive techniques required for generating either type of molecule, the need for extensive purification of heteroconjugates away from homoconjugates or mono-specific antibodies, and the generally low yields obtained.
Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions. A variety of recombinant methods have been developed for efficient production of BsAbs, both as antibody fragments (Carter et al., 1995, J. Hematother. 4(5): 463-70; Pluckthun et al., 1997, Immunotechnology 3(2): 83-105; Todorovska et al., 2001, J. Immunol. Methods 248(1-2): 47-66) and full length IgG formats (Carter, 2001, J. Immunol. Methods 248(1-2): 7-15).
Combining two different scFvs results in BsAb formats with minimal molecular mass, termed sc-BsAbs or Ta-scFvs (Mack et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92(15): 7021-25; Mallender et al., 1994, J. Biol. Chem. 269(1): 199-206). BsAbs have been constructed by genetically fusing two scFvs to a dimerization functionality such as a leucine zipper (Kostelny et al., 1992, J. Immunol. 148(5): 1547-53; de Kruif et al., 1996, J. Biol. Chem. 271(13): 7630-34).
Diabodies are small bivalent and bispecific antibody fragments. The fragments comprise a VH connected to a VL on the same polypeptide chain, by using a linker that is too short (less than 12 amino acid residues) to allow pairing between the two domains on the same chain. The domains are forced to pair intermolecularly with the complementary domains of another chain and create two antigen-binding sites. These dimeric antibody fragments, or “diabodies,” are bivalent and bispecific (Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90(14): 6444-48). Diabodies are similar in size to a Fab fragment. Polypeptide chains of VH and VL domains joined with a linker of between 3 and 12 amino acid residues form predominantly dimers (diabodies), whereas with a linker of between 0 and 2 amino acid residues, trimers (triabodies) and tetramers (tetrabodies) predominate. In addition to the linker length, the exact pattern of oligomerization seems to depend on the composition as well as the orientation of the variable domains (Hudson et al., 1999, J. Immunol. Methods 231(1-2): 177-89). The predictability of the final structure of diabody molecules is very poor.
Although sc-BsAb and diabody-based constructs display interesting clinical potential, it was shown that such non-covalently associated molecules are not sufficiently stable under physiological conditions. The overall stability of a scFv fragment depends on the intrinsic stability of the VL and VH domains as well as on the stability of the domain interface. Insufficient stability of the VH-VL interface of scFv fragments has often been suggested as a main cause of irreversible scFv inactivation, since transient opening of the interface, which would be allowed by the peptide linker, exposes hydrophobic patches that favor aggregation and therefore instability and poor production yield (Worn et al., 2001, J. Mol. Biol. 305(5): 989-1010).
An alternative method of manufacturing bispecific bivalent antigen-binding proteins from VH and VL domains is described in U.S. Pat. No. 5,989,830. Such double head and dual Fv configurations are obtained by expressing a bicistronic vector, which encodes two polypeptide chains. In the Dual-Fv configuration, the variable domains of two different antibodies are expressed in a tandem orientation on two separate chains (one heavy chain and one light chain), wherein one polypeptide chain has two times a VH in series separated by a peptide linker (VH1-linker-VH2) and the other polypeptide chain consists of complementary VL domains connected in series by a peptide linker (VL1-linker-VL2). In the cross-over double head configuration, the variable domains of two different antibodies are expressed in a tandem orientation on two separate polypeptide chains (one heavy chain and one light chain), wherein one polypeptide chain has two times a VH in series separated by a peptide linker (VH1-linker-VH2) and the other polypeptide chain consists of complementary VL domains connected in series by a peptide linker in the opposite orientation (VL2-linker-VL1). Molecular modeling of the constructs suggested the linker size to be long enough to span 30-40 Å (15-20 amino acid residues).
Increasing the valency of an antibody is of interest as it enhances the functional affinity of that antibody due to the avidity effect. Polyvalent protein complexes (PPC) with an increased valency are described in U.S. Patent Application Publication No. US 2005/0003403 A1. PPCs comprise two polypeptide chains generally arranged laterally to one another. Each polypeptide chain typically comprises three or four “v-regions,” which comprise amino acid sequences capable of forming an antigen binding site when matched with a corresponding v-region on the opposite polypeptide chain. Up to about six “v-regions” can be used on each polypeptide chain. The v-regions of each polypeptide chain are connected linearly to one another and may be connected by interspersed linking regions. When arranged in the form of the PPC, the v-regions on each polypeptide chain form individual antigen binding sites. The complex may contain one or several binding specificities.
A strategy was proposed by Carter et al. (Ridgway et al., 1996, Protein Eng. 9(7): 617-21; Carter, 2011, J. Immunol. Methods 248(1-2): 7-15) to produce a Fc heterodimer using a set of “knob-into-hole” mutations in the CH3 domain of Fc. These mutations lead to the alteration of residue packing complementarity between the CH3 domain interface within the structurally conserved hydrophobic core so that formation of the heterodimer is favored as compared with homodimers, which achieves good heterodimer expression from mammalian cell culture. Although the strategy led to higher heterodimer yield, the homodimers were not completely suppressed (Merchant et al., 1998, Nat. Biotechnol. 16(7): 677-81.
Gunasekaran et al. explored the feasibility of retaining the hydrophobic core integrity while driving the formation of Fc heterodimer by changing the charge complementarity at the CH3 domain interface (Gunasekaran et al., 2010, J. Biol. Chem. 285(25): 19637-46). Taking advantage of the electrostatic steering mechanism, these constructs showed efficient promotion of Fc heterodimer formation with minimum contamination of homodimers through mutation of two pairs of peripherally located charged residues. In contrast to the knob-into-hole design, the homodimers were evenly suppressed due to the nature of the electrostatic repulsive mechanism, but not totally avoided.
Davis et al. describe an antibody engineering approach to convert Fc homodimers into heterodimers by interdigitating β-strand segments of human IgG and IgA CH3 domains, without the introduction of extra interchain disulfide bonds (Davis et al., 2010, Protein Eng. Des. Sel. 23(4): 195-202). Expression of SEEDbody (Sb) fusion proteins by mammalian cells yields Sb heterodimers in high yield that are readily purified to eliminate minor by-products.
U.S. Patent Application Publication No. US 2010/331527 A1 describes a bispecific antibody based on heterodimerization of the CH3 domain, introducing in one heavy chain the mutations H95R and Y96F within the CH3 domain. These amino acid substitutions originate from the CH3 domain of the IgG3 subtype and will heterodimerize with an IgG1 backbone. A common light chain prone to pair with every heavy chain is a prerequisite for all formats based on heterodimerization though the CH3 domain. A total of three types of antibodies are therefore produced: 50% having a pure IgG1 backbone, one-third having a pure H95R and Y96F mutated backbone, and one-third having two different heavy chains (bispecific). The desired heterodimer can be purified from this mixture because its binding properties to Protein A are different from those of the parental antibodies: IgG3-derived CH3 domains do not bind to Protein A, whereas the IgG1 does. Consequently, the heterodimer binds to Protein A, but elutes at a higher pH than the pure IgG1 homodimer, and this makes selective purification of the bispecific heterodimer possible.
U.S. Pat. No. 7,612,181 describes a Dual-Variable-Domain IgG (DVD-IgG) bispecific antibody that is based on the Dual-Fv format described in U.S. Pat. No. 5,989,830. A similar bispecific format was also described in U.S. Patent Application Publication No. US 2010/0226923 A1. The addition of constant domains to respective chains of the Dual-Fv (CH1-Fc to the heavy chain and kappa or lambda constant domain to the light chain) led to functional bispecific antibodies without any need for additional modifications (i.e., obvious addition of constant domains to enhance stability). Some of the antibodies expressed in the DVD-Ig/TBTI format show a position effect on the second (or innermost) antigen binding position (Fv2). Depending on the sequence and the nature of the antigen recognized by the Fv2 position, this antibody domain displays a reduced affinity to its antigen (i.e., loss of on-rate in comparison to the parental antibody). One possible explanation for this observation is that the linker between VL1 and VL2 protrudes into the CDR region of Fv2, making the Fv2 somewhat inaccessible for larger antigens.
The second configuration of a bispecific antibody fragment described in U.S. Pat. No. 5,989,830 is the cross-over double head (CODH), having the following orientation of variable domains expressed on two chains:
VL1-linker-VL2, for the light chain, and
VH2-linker-VH1, for the heavy chain
The '830 patent discloses that a bispecific cross-over double-head antibody fragment (construct GOSA.E) retains higher binding activity than a Dual-Fv (see page 20, lines 20-50 of the '830 patent), and further discloses that this format is less impacted by the linkers that are used between the variable domains (see page 20-21 of the '830 patent).